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WO1998049325A1 - Proteines hybrides de marquage, vecteurs d'expression et lignees cellulaires transfectees de ces proteines destines a l'analyse du transport du noyau - Google Patents

Proteines hybrides de marquage, vecteurs d'expression et lignees cellulaires transfectees de ces proteines destines a l'analyse du transport du noyau Download PDF

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WO1998049325A1
WO1998049325A1 PCT/CA1998/000385 CA9800385W WO9849325A1 WO 1998049325 A1 WO1998049325 A1 WO 1998049325A1 CA 9800385 W CA9800385 W CA 9800385W WO 9849325 A1 WO9849325 A1 WO 9849325A1
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protein
gfp
proteins
reporter
nuclear
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Ursula Stochaj
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McGill University
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McGill University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/463—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from amphibians
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to reporter fusion proteins which allow the monitoring of facilitated transport and passive diffusion of proteins across the nuclear envelope in mammalian cells, cloning and expression vectors encoding these reporter proteins and transfected cell lines thereof.
  • NLSs nuclear . localization sequences
  • the nuclear pore complex mediates also diffusion, and proteins of molecular masses of 40kD or less can enter the nucleus via passive diffusion. By contrast, proteins that exceed the size of the diffu- sion channel enter the nucleus by a facilitated process that requires energy and a NLS. Polypeptides larger than 70kD are excluded from nuclei if they do not carry a functional NLS. NLSs can be divided into several classes. The NLS present in SV40 T-antigen is an exam- pie for a "simple" NLS containing a stretch of basic amino acid residues.
  • a second class of NLSs contains "bipartite" sequences that carry two clusters of posi- tively charged amino acid residues that are separated by a spacer region, such as the NLS of Xenopus laevis nucleoplasmin.
  • Another type of targeting sequences includes complex NLSs which do not fit into the classes of simple or bipartite signals.
  • GFP Aequorea victoria green fluorescent protein
  • SV40*-NLS mutant form of SV40-NLS SV40inv-NLS inverse sequence of SV40-NLS To analyze nucleocytoplasmic traffic in growing cells, an experimental system which is based on the inducible synthesis of fluorescent reporter proteins was developed. Reporter proteins of various sizes described here carry different NLSs which are fused to the Aequorea victoria green fluorescent protein (GFP). These substrates were expressed and localized in mammalian culture cells. In addition to the analysis of facilitated protein transport into nuclei, assays to study passive diffusion from the nucleus into the cytoplasm were described. To facilitate the generation of gene fusions that encode different mutants of GFP with or without a nuclear localization sequence several new cloning vectors were generated.
  • a stable cell line that produces a nuclear reporter protein under control of an inducible promoter was analyzed. Upon induction, single cells of this clone synthesize similar amounts of the reporter pro- tein.
  • such cell lines offer the advantage that large numbers of cells can be tested simultaneously under identical conditions. Additional stable cell lines are currently generated for more detailed analyses of transport and diffusion across the nuclear envelope. Taken together, novel experimental tools to investigate nucleocytoplasmic transport and diffusion in intact mammalian cells were described.
  • tagged nuclear transport substrates carrying various numbers of GFP to study nucleocytoplasmic traffic in vi tro were also generated. These substrates can be expressed in Escherichia coli and purified by metal affinity-chromatography . The purified substrates are currently tested with semi-permeabilized HeLa cells and with yeast spheroplasts to demonstrate that these proteins are bona fide substrates for nuclear transport.
  • genes encoding reporter proteins to monitor transport and passive diffusion of proteins across the nuclear envelope in mammalian cells which comprises a protein carrying a nuclear localization sequence (NLS) fused to at least one copy of a reporter protein, wherein the reporter protein allows for the visualization of the reporter fusion protein.
  • NLS nuclear localization sequence
  • the NLS derived from SV40 T-antigen is connected to the reporter protein via a linker sequence encoding small amino acid residues.
  • the NLS may also be derived from Xenopus laevis nucleoplasmin or present in the C-terminal portion of nucleoplasmin.
  • an expression vector to transiently and stably express reporter genes of the present invention which comprises a gene encoding a protein carrying a nuclear localization sequence (NLS) fused to at least one copy of a reporter protein, wherein expression of the gene is under the control of an inducible promoter.
  • NLS nuclear localization sequence
  • the NLS may be connected to the reporter protein via a linker sequence encoding small amino acid residues.
  • the reporter protein may be any fluorescent protein such as Aequorea vi ctoria green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • a cell line stably expressing a nuclear reporter protein to monitor trans- port and passive diffusion of proteins across the nuclear envelope, which comprises a cell line trans- fected by the expression vector of the present invention.
  • the NLS may be fused to the reporter protein by a linker encoding small amino acid residue.
  • the cell lines may be selected from the group consisting of HeLa, COS and CHO.
  • an in vivo assay for screening compounds which affect transport and/or passive diffusion of proteins across the nuclear envelope in mammalian cells which comprises the steps of: a) treating a stably transfected cell line of the present invention with a compound; and b) visualizing the distribution of reporter protein in nuclei and cytoplasm.
  • an in vi tro assay for screening compounds which affect transport and/or passive diffusion of proteins across the nuclear envelope in mammalian cells which comprises the steps of: a) treating semi-permeabilized cells which support in vitro nuclear transport with a compound and a pro- tein of the present invention; and b) visualizing the distribution of reporter protein in nuclei and cytoplasm.
  • the cells may be treated with the compound and protein concurrently or one after the other.
  • Fig. 1 illustrates a schematic representation of different fluorescent reporter proteins containing GFP used in this study
  • Fig. 2 illustrates transient expression of fusion proteins harboring one copy of GFP
  • Fig. 3 illustrates transient expression of transport substrates with two and four copies of GFP
  • Fig. 4 illustrates the effect of linker sequences on the localization of SV40-GFP4;
  • Fig. 5 illustrates the localization of reporter proteins harboring wild type and mutant versions of SV40-NLS
  • Figs. 6A and 6B illustrate nucleocytoplasmic diffusion of NP-GFP, (A) chilling of cells, (B) treatment with sodium azide/deoxyglucose at different temperatures ;
  • Fig. 7 illustrates the kinetics of the diffusion of NP-GFP across the nuclear envelope in HeLa cells treated with sodium azide/deoxyglucose
  • Fig. 8 illustrates stably transfected HeLa cells expressing nucleoplasmin-GFP.
  • an experimental system to study nucleocytoplasmic traffic of proteins in living mammalian cells.
  • substrates were generated that contain one or more copies of Aequorea victoria green fluorescent protein (GFP).
  • GFP Aequorea victoria green fluorescent protein
  • Reporter proteins of various sizes that carry different nuclear localization sequences (NLSs) were created to follow facilitated transport and passive diffusion across the nuclear envelope.
  • the expression of reporter genes was controlled by an inducible promoter.
  • Transiently and stably transfected HeLa cells were employed to follow the sorting of fluorescent reporter proteins.
  • NLS-GFP fusions were located in HeLa cells it was found that direct fusion of the NLS derived from SV40 T-anti- gen to GFP prevented nuclear accumulation of the protein.
  • insertion between NLS and GFP of different linkers encoding small amino acid residues produced reporter proteins that were competent for nuclear import.
  • a small protein of approximately 30kD that harbors one copy of GFP and the bipartite Xenopus nucleoplasmin NLS accumulates effi- ciently in nuclei of HeLa cells. Under conditions which abolish active facilitated nuclear transport the protein diffuses across the nuclear envelope.
  • This polypeptide can therefore be used as a reporter to analyze passive diffusion between nucleus and cyto- plasm.
  • histidine tagged versions of reporter proteins which permits their purification by metal affinity chromatography were also generated.
  • HtTA-1 cells are described in detail in Damke et al., 1995 (Damke, H. et al . , 1995, Meth . Enzym. ,
  • HtTA-1 cells contain the tTA-element which permits the control of gene expression by addi- tion of tetracycline to the growth medium.
  • HeLa cells were grown in Dulbecco's modified essential medium (DMEM) containing penicillin G (50U/ml), streptomycin (50 ⁇ g/ml) and 8 % fetal bovine serum (complete medium).
  • DMEM Dulbecco's modified essential medium
  • HtTA-1 cells were grown and transfected as described above. Transfected cells were grown under standard conditions for at least two weeks without selection. One day after addition of 50 nM dexametha- sone, cells were sorted by FACS into 24-well plates under sterile conditions . A maximum of one cell per well was obtained after sorting. Cells were further grown for two to three weeks and analyzed for the synthesis of nuclear reporter proteins. Towards this end, stable cell lines were kept in the presence of 50 nM dexamethasone ( 2 days ) and analyzed by fluorescence microscopy as described above. Plasmid constructions
  • Plasmid pGRE5-2 was used as a vector for genes encoding different fluorescent substrates. Gene expression is controlled by glucocorticoid response elements and can be induced by dexamethasone (Mader, S and White, J., 1993, Proc . Na tl . Acad. Sci USA, 90:5603- 5607). A mutant version of GFP in pBluescriptKS "1" carry- ing a Ser65 —> Thr65 substitution by PCR-amplification of the GFP-DNA using appropriate primers was generated. This created plasmid p580 which contains the mutant GFP allele cloned into the KpnI/EcoRI sites of pBluescript
  • Plasmids encoding nuclear substrates with one or two copies of GFP Plasmids encoding nuclear substrates with one or two copies of GFP
  • Nucleoplas- min-GFP i.e., the C-terminal half of Xenopus nucleo- plasmin fused to a single copy of GFP has been described (Chatterjee, S. and Stochaj , U., 1996, BioTechniques, 21: 62-63).
  • Plasmid p709 is derived from pGRE5-2 and codes for NP-GFP. Gene fusions containing two copies of GFP were created by removal of the UAG stop codon of GFP followed by insertion of a NotI linker (12mer, New England Biolabs). In addition, a Notl-linker was inserted into the KpnI-site of plasmid p580. Both copies of the GFP coding sequence were fused in frame via their Notl-sites thereby creating GFP2 in pBluescript (plasmid p690). Insertion of the KpnI/EcoRI fragment of p690 into the KpnI/EcoRI sites of pGRE5-2 created plasmid p785. For p785, the expression of GFP2 is driven by an inducible promoter carrying 5 glucocor- ticoid response elements. Plasmids encoding fusion proteins containing four copies of GFP
  • DNAs encoding different NLSs were cloned into pGRE5-2. These vectors were digested with Xhol and
  • DNA was digested with Xhol and
  • Agel Recessed ends were filled in and DNA was autoli- gated.
  • Derivatives of plasmids carrying wild type or mutant versions of SV40- NLS fused to GFP4 were generated by in frame insertion of linker sequences. Constructs encoding a direct fusion of NLS and GFP were digested with Agel, incubated with T4-polymerase in the presence of 4 dNTPs.
  • Linker insertion generated the following additional amino acid residues between NLS and the first copy of GFP: BamHI ala-asp- pro-pro (ADPP), EcoRI gly-asn-ser-pro (GNSP), Smal ala- pro-gly-pro (APGP).
  • ADPP BamHI ala-asp- pro-pro
  • GNSP EcoRI gly-asn-ser-pro
  • APGP Smal ala- pro-gly-pro
  • Protein SV40-GFP4 that contains the linker sequence ala-asp-pro-pro is referred to as SV40- ADPP-GFP4. All other constructs are named accordingly.
  • Plasmid p713 encodes GFP4 whose expression is controlled by five glucocorticoid response elements. To obtain this plasmid a KpnI/EcoRI fragment coding for the in frame fusion of four copies of GFP was inserted into the KpnI/EcoRI sites of
  • Plasmids to generate gene fusions containing GFP or NP- GFP To facilitate the generation of gene fusions containing different forms of GFP the following constructs were generated: Plasmid p827 was obtained by inserting a Xbal/PstI fragment encoding a mutant GFP (L64 T65) into the Xbal/PstI sites of pKS + . p657 is derived from p580; p580 was digested with BsmI and treated with T4-DNA-polymerase in the presence of 4 dNTPs. A Notl-linker (12mer, New England Biolabs) was ligated to the filled in DNA, followed by digestion with NotI and autoligation.
  • p657 is used to create fusion genes that encode GFP at their 5 '-end.
  • a derivative of p657 was generated by inserting the Ncol/Hpal fragment of p827 into p657 that was treated with Ncol and Hpal .
  • the resulting plasmid p833 contains GFP (L64 T65) carrying a Notl-linker at the former Bsml-site.
  • p834 was created by insertion of the
  • Plasmids encoding histidine-tagged versions of fluorescent marker proteins To generate genes encoding histidine-tagged marker proteins, coding sequences for the various fluorescent substrates were inserted in the appropriate pQE vector (Qiagen) to create in frame fusions of the histidine tag and the reporter protein. Specifically, to obtain His6-GFP4, plasmid p713 was digested with Agel and EcoRI, blunted and inserted into pQEll which was treated with BamHI and Klenow polymerase. These manipulations created plasmid p779 which encodes His6- GFP4 under control of the Escherichia coli tac pro- moter.
  • Plasmid p779 was digested with NotI, the 4.8kb fragment was autoligated resulting in plasmid p788 which encodes His6-GFP2. Treatment of plasmid p779 with Hpal and autoligation of the 4. lkb linearized band generated plasmid p790 which carries the coding sequence for His6-GFP1. To generate His6-NP-GFP4 , a Hindlll/EcoRI fragment containing the coding sequence for NP-GFP4 was blunted and cloned into pQElO treated with BamHI and Klenow-polymerase to remove recessive ends. Plasmid p778 codes for protein His6-NP-GFP4.
  • plasmid p791 which encodes His6- NP-GFP2.
  • Digestion of plasmid p778 with Hpal and autoligation of the 4. lkb band results in plasmid p792 which codes for His6-NP-GFP.
  • the respective pGRE5-2 vectors containing the constructs described were digested with EcoRI and Hindlll followed by treatment with T4 DNA-polymerase in the presence of 4 dNTPs.
  • GFP L64 T65 This mutant GFP-derivative GFPmutl, referred to as GFP L64 T65, was obtained from B. R. Cormack and published in 1996 ( Gene, 174:33-38).
  • nuclear substrates carrying the blue version of GFP are being created.
  • the blue version of GFP is mutant P4 obtained from Roger Tsien (Heim, R. et al . , 1994, Proc . Natl . Acad. Sci . , USA, 91:12501-12504). Plasmids for expression in Saccha.rom.yces cerevlslae
  • Genes encoding different nuclear substrates described in Table 1 are presently cloned into yeast expression vectors. The expression of gene fusions in these vectors is driven by the GAL1 control region. Gene expression is therefore induced when cells are grown on galactose as carbon source.
  • Fig. 1 Controls include proteins with one, two and four copies of GFP, which are termed GFP, GFP2 and GFP4, respectively. Fusion proteins were generated that carry the nucleoplasmin bipartite NLS (NP), the C- terminal half of nucleoplasmin (nucleoplasmin), SV40 wild type NLS (SV40), a mutant derivative (SV40*) and the inverse signal (SV40inv). Additional fusions were created that contain linker sequences between the NLS and the first copy of GFP. Only one construct harboring a linker sequence L and SV40-NLS is shown as an example. The different linker sequences inserted and fusion proteins containing these linkers are described below.
  • Control proteins include GFP and a fusion protein containing two and four copies of GFP, termed GFP2 and GFP4, respectively.
  • Polypeptide Nucleoplasmin- GFP contains the C-terminal half of Xenopus nucleoplasmin including the bipartite NLS and the DNA- binding region.
  • Proteins SV40-GFP4, SV40*-GFP4, SV40inv-GFP4 and NP-GFP4 carry wild type or mutant forms of the NLS at their N-termini as indicated. Derivatives of the proteins containing different linker sequences between NLS and GFP4 are described above.
  • the estimated molecular masses of GFP2 is about 60kD, for GFP4 and the NLS-containing derivatives approximately 120kD. When synthesized in E. coli and analyzed by denaturing gels, proteins migrate as expected.
  • HeLa cells were transiently transfected with plasmids that express fusion proteins under the control of an inducible promoter. Gene expression was induced by addition of dexamethasone to the growth medium. As shown in Fig. 2 for plasmids encoding NP-GFP and GFP, in the presence of dexamethasone cells exhibit bright fluorescence. HeLa cells were transiently transfected with plasmids encoding NP-GFP (panels A, B) or GFP (panels C, D) and gene expression was induced with dexamethasone as described herein.
  • NP-GFP is efficiently targeted to the nucleus due to the presence of the bipartite NLS derived from Xenopus nucleoplasmin.
  • non-transfected cells display only a low level of autofluorescence (Fig. 2).
  • Low autofluorescence signals were also obtained for non-induced cells, i.e., cells kept in the absence of dexamethasone.
  • linker insertion generated altered nuclear substrates that are recognized by the nuclear transport apparatus and targeted to the nucleus.
  • linker insertions were created for SV40*-GFP4 and SV40inv-GFP4, and we analyzed the cellular localization of the respective proteins (Fig. 5).
  • GFP4-derivatives containing the linker sequence ADPP (Panels A to F) or APGP (panels G to L) were transiently synthesized in HeLa cells.
  • Substrate proteins contained wild type SV40-NLS (panels A, B, G, H), mutant SV40*-NLS (panels C, D, I, J) or inverse SV40inv (panels E, F, K, L). Staining with DAPI (panels A, C, E, G, I, K) and green fluorescence (panels B, D, F, H, J, L) is shown.
  • Transport substrates carrying SV40inv-NLS were excluded from the nucleus (Fig. 5).
  • proteins containing the mutant SV40*-NLS were not excluded from nuclei.
  • these reporter proteins did not accumulate in nuclei.
  • NLS nuclear transport substrates containing four copies of GFP and different NLSs.
  • the type and context of the NLS specifies the localization of the reporter protein that can be easily followed in living cells. Diffusion from the nucleus into the cytoplasm
  • NP-GFP i.e., a fusion protein that contains the Xenopus nucleoplasmin NLS fused to one copy of GFP accumulated in nuclei under normal growth conditions
  • NP-GFP small size of approximately 30kD should permit NP-GFP to also traverse the nuclear pore complex by passive diffusion.
  • transiently transfected cells synthesizing NP-GFP showed bright nuclear fluorescence (Fig. 2).
  • the inducer dexamethasone was removed and cells were transferred to 4°C. Although this led to diffusion of NP-GFP from the nucleus into the cytoplasm, even after 4 hours of incubation at 4°C the nuclear fluorescence was more intense than the fluorescence signal seen for the cytoplasm.
  • NP-GFP sodium azide/2-deoxy D-glucose
  • azide/deoxyglucose sodium azide/2-deoxy D-glucose
  • Fig. 7 HeLa cells were incubated at room temperature with sodium azide/deoxyglucose and the nucleocytoplasmic distribution of the fluorescence signal was determined for 0 min, 10 min, 30 min, 45 min, 60 min and 90 min of the incubation with sodium azide/deoxyglucose as indicated in Fig. 7.
  • transiently transfected cells When induced with dexamethasone, transiently transfected cells show differences in the fluorescence which reflects different copy numbers of the plasmids introduced into each cell. In addition, transiently transfected cells loose the plasmid after prolonged culturing. Therefore, for many studies it is advantageous to have a uniform population of cells with comparable fluorescence. To this end, we created a stable cell line which carries the gene for nucleoplasmin-GFP. Cells were transfected and grown for two weeks without selection. Dexamethasone was added to the growth medium and cells displaying fluorescence were sorted by FACS . Under these conditions, approximately one in 10,000 cells gave a fluorescence signal. Single cells were isolated and further cultured.
  • NLS-GFP derivatives Different plasmid vectors encoding NLS-GFP derivatives will be useful to create fusion proteins that are targeted to the nucleus. If the protein of interest has deleterious effects, it is desirable to have an experimental system that permits the controlled induction of genes. To this end, promoters regulated by steroid hormone binding sites as described here for pGRE-derivatives will be optimal.
  • flanking sequences pro- vided by GFP could interfere with its function. It is currently believed that nuclear transport of proteins harboring SV40-NLS and the bipartite nucleoplasmin-NLS are both recognized by importin-alpha during nuclear transport. Since nucleoplasmin NLS directly fused to GFP or GFP4 was efficiently targeted to the nucleus, it is reasonable to assume that the N-terminal sequence of GFP does not inhibit binding to cytoplasmic NLS-recep- tors in general. We therefore favor the interpretation that SV40-NLS is not readily accessible for recognition by nuclear transport factors when directly fused to GFP4.
  • the small size of GFP permits this protein to enter the nucleus by passive diffusion. Fusion of the bipartite nucleoplasmin NLS to GFP efficiently mediated nuclear accumulation of the reporter protein.
  • the small size of NP-GFP i.e., approximately 30kD, made it an ideal tool to study diffusion from the nucleus into the cytoplasm.
  • NP-GFP exited the nucleus at low tempera- tures even in the absence of metabolic inhibitors.
  • Migration of NP-GFP into the cytoplasm of HeLa cells during chilling is not caused by irreversible damage of the cells or the nuclear envelope. HeLa cells survived this treatment, and the nuclear substrate was re- imported into nuclei after shifting to normal growth conditions.
  • the rapid nuclear exit of NP-GFP during chilling enables us to study diffusion in the absence of metabolic inhibitors which are likely to interfere with a variety of cellular processes.
  • NP- GFP will be useful as a tool to test how various drugs might interfere with diffusion across the nuclear envelope. The possible effect by these agents can be easily followed by fluorescence microscopy. Stable cell lines expression fluorescent reporter protein
  • NP-GFP or only GFP have also been generated, and we are currently creating additional cell lines which have improved fluorescence .
  • all of the cell lines described will allow the controlled expression not only of the fluorescent reporter protein but also of additional genes of interest. Since the cells contain the tTA-element (Damke, H. et al . , 1995, Meth . Enzym. , 257:209-220), they can be used to regulate the synthesis of a protein of interest by addition of tetracycline to the growth medium. Genes encoded on a tetracycline response plasmid are repressed in the presence of tetracycline, whereas in the absence of tetracycline gene expression is induced.
  • Nuclear substrates described in this study were also tested in other mammalian cell lines such as COS-7 and CHO cells. Similar results as described for HeLa cells were obtained, indicating that the reporter system we have developed is useful as a model to study nuclear transport in different mammalian cells. Taken together, we have established tools to study diffusion and signal-mediated transport across the nuclear envelope in higher eukaryotic cells. This sets the stage to analyze nucleocytoplasmic traffic in living mammalian cells in more detail.
  • Histidine-tagged (His6) versions of fluorescent reporter proteins have been created. Synthesis of these proteins is inducible in Escherichia coli . Fusion proteins are currently purified by affinity-purifica- tion and analyzed in in vi tro nuclear transport assays. The advantage of these substrates is that they are easy to purify in large quantities and at low costs. Previously used substrates for in vi tro studies involved the synthesis and covalent coupling of NLS- peptides, an expensive and laborious procedure.

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Abstract

La présente invention se rapporte à des protéines hybrides marqueurs qui permettent le contrôle du transport facilité et de la diffusion passive de protéines à travers la membrane nucléaire de cellules de mammifères; au clonage et à des vecteurs d'expression codant ces protéines marqueurs et les lignées cellulaires transfectées de ces protéines. La présente invention concerne plus particulièrement des gènes codant pour des protéines marqueurs en vue de contrôler le transport et la diffusion passive de protéines à travers la membrane nucléaire de cellules de mammifères qui comporte une protéine portant une séquence de localisation du noyau (NLS) qui a fusionné en au moins une copie d'une protéine marqueur. La protéine marqueur permet la visualisation de la protéine hybride marqueur.
PCT/CA1998/000385 1997-04-28 1998-04-24 Proteines hybrides de marquage, vecteurs d'expression et lignees cellulaires transfectees de ces proteines destines a l'analyse du transport du noyau Ceased WO1998049325A1 (fr)

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Cited By (4)

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WO2002018572A3 (fr) * 2000-08-25 2003-09-12 Aventis Pharma Inc Peptides de penetration de membrane et utilisations associees
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WO2016064742A1 (fr) * 2014-10-21 2016-04-28 The Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University Of Nevada, Las Vegas Procédés et compositions permettant le dépistage de fonction moléculaire comprenant des mini-motifs chimériques

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WO2002018572A3 (fr) * 2000-08-25 2003-09-12 Aventis Pharma Inc Peptides de penetration de membrane et utilisations associees
US7754678B2 (en) 2000-08-25 2010-07-13 Aventis Pharmaceuticals Inc. Membrane penetrating peptides and uses thereof
WO2003044535A1 (fr) * 2001-11-22 2003-05-30 Mehmet Ozturk Methode, antigene et anticorps permettant de distinguer des cellules apoptotiques et viables
WO2016064742A1 (fr) * 2014-10-21 2016-04-28 The Board Of Regents Of The Nevada System Of Higher Education On Behalf Of The University Of Nevada, Las Vegas Procédés et compositions permettant le dépistage de fonction moléculaire comprenant des mini-motifs chimériques

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